An overview on dispersion procedures and testing methods for the ecotoxicity testing of nanomaterials in the marine environment

NMs dispersion

• NMs ecotoxicity to marine organisms was reviewed, focusing on methodological issues.• <5 % of studies used a standard protocol for NMs dispersion.• >60 % combined a non-standard dispersion method with NMs characterization.
• MeOx were the most studied NMs, but interest is growing on nanoplastics and MCNMs.• Primary producers were the most investigated marine species.
species.However, the efforts to characterize NMs in ecotoxicological media recognize the importance of following conditions that are as standardized as possible to support the ecological hazard assessment of NMs.

Introduction
The roadmap outlined by the European Green Deal requires that any new material or product should be not only functional and cost-effective but also safe and sustainable to ensure compliance with regulation and acceptance by consumers (Gottardo et al., 2021).Therefore, a comprehensive risk assessment of new materials/products, investigating both human and environmental exposure, is essential, especially for highly complex advanced materials, defined as materials rationally designed to have new or enhanced properties and/or targeted or enhanced structural features that improve functional performance over conventional products and processes (Kennedy et al., 2019; Organisation for Economic Co-operation and Development (OECD), 2022).Many engineered nanomaterials (NMs) fall in this category, among which the multicomponent nanomaterials (MCNMs), i.e., materials having two or more nano-components held together by strong molecular bonds or other physico-chemical forces, or nanomaterials modified by hard or soft coatings (Saleh et al., 2015).
Since both single and MCNMs are contained in various commercial products and materials, they can be released and reach natural waters through different routes, such as wastewater discharge, river influx, runoff from agricultural and urban areas, and atmospheric deposition (Geitner et al., 2020).Thus, understanding the fate and behaviour of NMs in aquatic systems is of critical importance, especially considering that, ultimately, the marine environment may represent the potential sink for these materials, which can enter coastal systems also through directs sources such as NM-containing personal care products (cosmetics and sunscreens) and antifouling paints for vessel hulls (Gondikas et al., 2020;Matranga and Corsi, 2012).
Based on the currently available literature, it has been recognized that the fate and behaviour of NMs in the aquatic environment is governed by complex processes, largely determined by the intrinsic properties of the different types of NMs and by the characteristics of the water system in which they are dispersed, including aggregation, agglomeration, sedimentation, dissolution, redox and photochemical reactions, as well as interactions with biological components (Lead et al., 2018;Turan et al., 2019;Zhang et al., 2022).In marine ecosystems, aggregation and particle settling usually occur at a faster rate than in freshwater environment due to the higher ionic strength and the lower concentration of natural organic matter (NOM).Together with pH, these parameters have been identified as relevant factors affecting the colloidal stability of NMs in surface waters (Badetti et al., 2021;Brunelli et al., 2022Brunelli et al., , 2018;;Klaine et al., 2008;Oomen et al., 2014).
All the transformation and transport processes listed above may influence the bioavailability and the potential effects of NMs to aquatic organisms in the environment and, consequently, are likely to affect also the outcome of ecotoxicological testing, especially when conducting bioassays with saltwater species.
Originally, the ecotoxicity of NMs was assessed using guidelines and protocols developed for chemicals, which were applied without considering all the potential modifications these materials may undergo in the different aquatic media employed.Afterwards, based on the increasing knowledge acquired on NMs fate and behaviour, a thorough investigation of these processes through the recently developed analytical techniques was deemed of primary importance for a thorough evaluation of their toxicity, as well as for cross-comparison of data from different studies (Mourdikoudis et al., 2018;Tantra et al., 2011).Testing NMs ecotoxicity was considered challenging also from the practical perspective, highlighting the need for the identification of suitable test conditions and potential modifications of testing protocols (Handy et al., 2012).For these reasons, the use of standardized methods and guidelines allowing to obtain reproducible measurements are highly recommended to ensure comparable high quality ecotoxicological data, and appropriately support the ecological risk assessment of NMs (Hartmann et al., 2015).
From the early 2000's, numerous efforts have been devoted to improve/modify the existing guidelines and protocols on chemicals for NMs.Regarding sample preparation for ecotoxicity testing, different dispersion protocols have been proposed (e.g., PROSPEcT, US CEINT/ NIST 1200 series, NANoREG-ECOTOX) with the aim to obtain as homogeneous and stable dispersions as possible along the assay duration.In 2006, the OECD established the Working Party on Manufactured Nanomaterials (as part of the Programme on Safety of Manufactured Nanomaterials), recognizing the importance of adapting existing OECD ecotoxicity test methods to NMs and developing specific guidelines and documents.Among these, the most recent ones are: i) Test Guideline -Dispersion stability of nanomaterials in simulated environmental media (OECD, 2017); ii) Guidance document 317 -Guidance document on aquatic and sediment toxicological testing of nanomaterials (Organisation for Economic Co-operation and Development (OECD), 2020a); iii) Guidance document 318 -Guidance document for the Testing of Dissolution and Dispersion Stability of Nanomaterials, and the Use of the Data for Further Environmental Testing and Assessment (OECD, 2020b, revised in July 2021).In particular, Guidance documents 317 and allowed to fast progress in a better determination of environmental hazard and behaviour testing of NMs: Guidance document 317 focuses on critical issues such as the choice of the medium, the exposure metrics and whether to include NMs that settle during the experiment in the exposure assessment for water-only bioassays, while Guidance document 318 gives detailed advice on how to apply existing test guidelines, or modify them for NMs, and how to report and interpret the results (Petersen et al., 2021).
Recommendations for environmental NMs testing media harmonization (i.e., five broad categories of testing media) were proposed by Geitner et al., 2020, who suggested a minimum set of parameters to measure for each saline medium type, including pH, ionic strength, dissolved organic matter (as primary parameters, essential for minimum characterization), key nutrients and particulate matter (as secondary ones, i.e. highly desirable).However, most ecotoxicological protocols for NMs focus on freshwater bioassays (Hund-Rinke et al., 2016), emphasising the need to further identify reference materials and species that are representative of marine ecosystems (Selck et al., 2016).
In this context, the main aim of this review is to provide an overview of the studies and advancements on the dispersion as well as exposure procedures to assess NMs ecotoxicity in the marine environment achieved by the scientific community from 2010.A particular attention has been devoted to the available standard dispersion protocols and technical guidelines to perform an ecotoxicological assay, investigating whether, in addition to the adoption of a specific dispersion procedure, an in-depth physico-chemical characterization of NMs in the ecotoxicological media was performed.Furthermore, information on the type of NMs studied, the marine organisms tested and the exposure procedures used, was gathered and comprehensively analysed.

Standard dispersion protocols and guidelines for ecotoxicological testing of nanomaterials
The selection of a dispersion procedure for (eco)toxicological testing is always a trade-off between not altering the properties of the tested material related to its functionality and, at the same time, obtaining a dispersion as homogeneous and stable as possible over time, to ensure the exposure dose selected for the experiments (Callaghan and MacCormack, 2017;Hartmann et al., 2015).Moreover, any additional component (e.g., dispersing agents as Tween 20, bovine serum albumin or natural organic matter) should be carefully considered according to the aim of the testing.
In the past decade, many efforts have been devoted to develop reliable, reproducible and relevant methods for testing and assessing the effects of NMs on human health and the environment in a regulatory context.Hereafter we consider a dispersion protocol as standard when it is developed from principles and guidelines from international standards organizations (according to the PROSPEcT by Nanotechnology Industries Association, 2010 definition).On the other hand, if no standard protocols are followed, we referred to dispersion methods.
As reported by Hartmann et al., 2015,  Based on the information included into these protocols, further ones have been developed purposely for ecotoxicological testing of NMs.On chronological order, a first protocol was released in 2010 within the PROSPEcT Programme (Nanotechnology Industries Association, 2010).At the same time, starting from the first draft of the NANOGENOTOX protocol (Jensen et al., 2011), the NANoREG-ECOTOX dispersion protocol was released in the context of the NANoREG EU Project (Booth and Jensen, 2015).In parallel, the CEINT/NIST US collaboration led to a protocol for the preparation of TiO 2 NPs in an environmental matrix for eco-toxicological assessment (Taurozzi et al., 2013).Besides these protocols, OECD proposed a technical guideline (TG 318) to assess the ability of a NM to attain a colloidal dispersion in simulated environmental media (OECD, 2017).This guideline is crucial since information on NMs dispersion stability, agglomeration behaviour and dissolution rate are the key prerequisites for a robust and reliable testing of NMs.Following this TG, NMs can be then categorised into three different stability classes, by determining their dispersion stability through UV-Vis spectroscopy in aqueous media with different ranges of pH, divalent ions and NOM concentration.
Based on the constant evolution of protocols over the years, those developed for ecotoxicological tests using the most widely available NMs on the market are summarized in Table 1, along with the TG 318.Key operating conditions, i.e., the NMs used in protocol development, the stock dispersion concentration, the pre-wetting of powder particles, the time and type of sonication, the power setting, the delivered sonication energy, the use of dispersing agents, the maximum time of stability assured and quality assurance, are reported and discussed.
Given the intrinsic complexity of the NMs, each protocol listed in Table 1 was developed for specific NMs or NMs' classes, where metal oxides were the most NMs tested.This is probably due to both their extensive use in various commercial products and their high tendency in agglomerating and settling after dispersion in aqueous media.In addition to the protocols in Table 1, the TG 318 expanded the range of NMs considered, developing a dispersion guideline that can be applied to all NMs with density > 1 kg/L (e.g., polymer-based NMs are excluded).
Regarding the stock dispersion concentration, PROSPEcT and NANoREG-ECOTOX suggest almost the same operating conditions, while a lower stock concentration is indicated by CEINT/NIST (1200-5).A. Brunelli et al.In the approach proposed by the TG 318, the focus lies on the particle number concentration range, suggesting not to exceed 20 times the concentration of the sample to be analysed.The NMs pre-wetting by EtOH at 0.5 % (v/v) is mentioned by PROSPEcT and NANoREG-ECOTOX, while CEINT/NIST does not specify any pre-wetting treatment.The TG 318 suggests using ultrapure water and leaving the NMs in the form of wet-paste for 24 h to ensure the proper interaction of NM surface with water.As concerns the type of sonication, all the protocols and the TG 318 listed in Table 1 refer to probe sonication.In addition, for high aspect ratio NMs, the TG 318 recommends performing sonication carefully to avoid breakage.
Moving from the selection of the technical equipment to the power input, the sonication time and the delivered sonication energy (DSE), some similarities but also differences among the examined protocols and TG 318 are observed.All these procedures suggest to perform calorimetric measurements to obtain the power input value (P) of the specific sonicator used.Then, PROSPEcT and CEINT/NIST report a fixed sonication time for each NM used to test the protocol, while NANoREG-ECOTOX and the TG 318 suggest determining this parameter experimentally, by increasing the sonication time and checking the hydrodynamic diameter of NMs to find the best fit between the lowest sonication time and the lowest NMs agglomeration.The output of this second approach, also described by other recent protocols for toxicity testing (DeLoid et al., 2017;Kaur et al., 2017), is the calculation of DSE.However, to date there is still a strong debate within the scientific community on the DSE to be used, which implies to specify the power input, the sonication time and the volume used, as well as on the stabilizing agents to be or not to be added.In particular, open issues concern: i) the use of a predefined DSE instead of determining a material-specific critical sonication energy (DSE cr ); ii) whether or not to use a stabilizing agent (such as NOM), which could interfere in the ecotoxicity assay by producing a different response with respect to the potential effects observed with NMs alone.
Concerning the stabilizing compounds, humic acid or Suwannee River NOM (a standardized and purified surface water NOM from the International Humic Substance Society) have been included in all the protocols and TG 318 listed in Table 1, except for the PROSPEcT protocol, which does not specify any dispersant.Regarding the maximum time of stability, given the quite intrinsic good stability of TiO 2 NMs in water and the corresponding relatively low stock concentration, the protocol that should guarantee the longer dispersion stability is the CEINT/NIST 1200-5, i.e., 48 h, while PROSPEcT and NANoREG-ECOTOX should work within one hour.The TG 318 does not guarantee a maximum time of stability but rather recommends assessing it after 6 h.All the ecotoxicological protocols suggest checking at least the hydrodynamic size over time by DLS.The TG 318 also indicates to follow agglomeration behaviour and settling of NMs by UV-Vis spectroscopy.
Overall, the TG 318 takes advantage of the protocols' development over the years by providing a more general approach that allows to select the best conditions for each NM type.In fact, considering the wide variety of NMs with unique/new characteristics that can influence their colloidal behaviour, it is extremely challenging to develop a general dispersion protocol that can be applied to all different categories of NMs.Among the critical factors listed in Table 1, which may contribute to the transformation processes of NMs (i.e.dissolution/leaching, aggregation/agglomeration, degradation) and thus influence their colloidal stability, it is possible to list some key elements to take into consideration.In particular, DSE should be calculated and clearly specified in any laboratory experiment since it can highly affect the generation of artifacts (Petersen et al., 2014).Indeed, the extreme localized temperature and pressure generated by the cavitation process can lead to the formation of highly reactive species within the stock dispersion (Mason and Peters, 2002), thus changing the chemical and/or physical stability of the system.Moreover, if the addition of any dispersant such as NOM or specific organic molecules such as alginate is required within the stock dispersion, the formation of potential byproducts or a unique layer structure termed as eco-corona has to be verified, since they can affect the assays results (Liu et al., 2022;Natarajan et al., 2021).It has also been demonstrated that, in the presence of NOM, the original NMs toxicity can be decreased since NOM is able to enhance NMs agglomeration by changing the surface potential (Nigro et al., 2021;Saavedra et al., 2019) or to form complexes with toxic ions released from NMs, thus reducing their final available concentration (Ouyang et al., 2018;Wang et al., 2015).Also, NOM may hinder the direct interaction between NMs and tested organisms (Li et al., 2021) or relieve oxidative damage by scavenging extracellular reactive oxygen species (Su et al., 2013).As far as the sonicator probe is concerned, it should be ensured that no particles are generated from its surface, and ice bath is always recommended to mitigate temperature increase or particle sintering during sonication.Finally, if strictly needed, the mixing of dispersants (e.g., surface coatings, NOM) with NMs should be done only after sonication to avoid artifacts and, in addition, a control test without NMs should be carried out to exclude possible adverse effects caused by the medium components (Petersen et al., 2014).
After adopting a dispersion protocol, the next step is the dilution of the stock dispersion in the ecotoxicological medium according to the concentrations of NMs needed for the assays.This could lead to variations in the behaviour of NMs (e.g., dissolution, agglomeration and sedimentation, eco-corona formation), depending on their interactions with the organic and inorganic components of the medium used, e.g.(macro)molecules and ions, at the selected concentration range.In this regard, the TG 318 (in the revised version of July 2021) lists the concentration ranges of the major representative natural water components (i.e., Na + , Ca 2+ , Mg 2+ , NO 3− , SO 4 2− , Al 3+ and natural dissolved organic matter) which can affect the NMs' stability in different aqueous media.Synthetic water can be obtained by opportunely combining these components, which were set to account for 95 % of the conditions found in natural waters.Moreover, TG 318 also indicates the effects (e.g., stabilization/destabilization) and the corresponding strength (i.e., low, medium, high), together with the relative abundance of these compounds in natural waters.Focusing on the marine environment, it is known that the high ionic strength of marine water is able to compress the electric double layer of the NMs surface, favouring agglomeration/aggregation and sedimentation processes (Amal et al., 1992;Chen and Elimelech, 2007), which are not counterbalanced by the relatively low amount of dissolved natural organic matter present.As a result, the tested species could be exposed to different NMs concentrations compared to the nominal ones, due to the possible formation of a concentration gradient.Therefore, the physico-chemical characterization of the NMs in the exposure medium, as well as the characteristics of the target species, become of utmost importance in the design of ecotoxicological assays for marine organisms, and in the interpretation of results.

Data collection and management
A systematic literature search was carried out on the Scopus database (Elsevier) from 2010 to October 2023 searching in the title, abstract and keywords "Nanoparticle OR Nanomaterial" AND "Ecotoxicity OR Ecotox" AND "Marine OR Sea water".This search produced 214 publications, reduced to 89 as follows: i) review papers were excluded; ii) only experimental studies using in vivo exposure were considered; iii) studies in which NMs were not dispersed in water media were excluded (e.g., NMs added in agar, in sediment, in feed).
The main information extrapolated from all the 89 scientific works is reported in Table 2, according to: • Tested NMs; • Marine organisms used as test species, and bioassay experimental set up; • Information on the NMs dispersion procedures, considering the method (i.e., sonication/vortexing/agitation), the medium, and the

Table 2
Ecotoxicological studies selected through the literature search, considering i) the tested NMs; ii) the tested organisms; iv) the dispersion procedure, including the method (i.e., sonication (S), bath-sonication (BS), probe sonication (PS), vortexing (V), agitation (A)), the medium and the duration for the stock dispersion preparation; iii) the exposure procedure, based on the exposure method (i.e., static or semi-static, which indicate a change of the medium at a certain time), the medium and the duration of the ecotoxicological assay; nr: conditions not reported.time used to prepare the stock dispersion, highlighting whether a standardized protocol was followed; • Information on the exposure procedure, considering if the ecotoxicological testing was conducted under static or semi-static conditions (i.e., medium not renewed and renewed during the experiment, respectively), the test medium used (e.g., Artificial Sea Water or Filtered Natural Sea Water) and the exposure time.
In parallel to Ag-based NMs, TiO 2 -based NMs have been employed for many years and investigated for their effects towards marine organisms by different authors (15 % of the total), starting from the uncoated ones (Barmo et al., 2013;Broccoli et al., 2021;Huang et al., 2016;Libralato et al., 2013;Minetto et al., 2017;Zhu et al., 2011a) to those coated with different molecules or polymers (e.g., -COOH, -NH 2 , PEG) or tested in combination with other chemicals (Balbi et al., 2014;Deng et al., 2022).These surface modifications aim at improving the performances of the final product, but they can also affect the impacts of these NMs on marine organisms (Connolly et al., 2022).The widespread use of TiO 2 -based NMs in different products is mainly related to their photocatalytic properties, finding application in self-cleaning paints (Amorim et al., 2018), self-sterilizing surfaces (Khan and Malik, 2022), cements for building façades (Fernandes et al., 2020) and asphalts for NO x abating (Fan et al., 2018).TiO 2 -based NMs also found an extensive usage in cosmetics such as sunscreens (Chaki Borrás et al., 2020), from which they can enter to the sea both: i) directly, e.g., from sunscreensreaching concentrations of μg/L during the summer season (Slomberg et al., 2021) -or from anti-fouling paints for boat protection, or ii) indirectly, e.g.entering from urban and industrial sewage.
b Alginic acid: use as a source of dissolved organic carbon.
configurations and the high antimicrobial effectiveness of ZnO-and CuO-based NMs led to their widespread use in many consumer products, such as protective and antifouling paints and varnishes, accounting for the assessment of their ecotoxicity versus marine species by many researchers (12 and 7 % of the total, respectively), e.g., by comparing the effects on marine organisms of CuO NMs, uncoated or coated with PEG, -COOH and -NH 2 (Connolly et al., 2022a) or of ZnO NMs with different shapes, such as spheres and nanorods (Dobretsov et al., 2020;Peng et al., 2011).Then, the last class of metal oxide NMs tested with marine organisms in 8 % of total studies, includes different types, among which CeO 2 -based NMs, namely CeO 2 -alginate and CeO 2 -chitosan, were the most studied ones (Nigro et al., 2021;Villa et al., 2020).
In addition to Ag and metal oxides NMs, the studies focusing on adverse effects of metal-based NMs towards marine species are 15 % of the total, with the Au-based and quantum dots as the most represented ones.Within this class of materials, two studies, one dealing with the effects caused by the interaction between Au NMs and microplastics (Davarpanah and Guilhermino, 2019) and one with engineered aminoclays (Choi et al., 2014), were also included.
Despite the unique mechanical properties and the outstanding electrical and heat conductivity of carbon-based NMs, these materials were studied only by a few authors (5 % of the total).This could be attributed to the difficulty of preparing a stable dispersion over the duration of the assay and the challenges related to their physico-chemical characterization by the most widespread analytical techniques, e.g., dynamic light scattering.
Advanced materials such as MCNMs are being used more frequently in novel products, by exploiting the new properties given by the synergistic benefits of the different components or by enhancing the existing ones.It is worth noting that, due to these intrinsic complexity and dynamic behaviour of these materials, new information on their hazard profiles is needed.Therefore, studies on the effects of MCNMs towards marine species are increasing in number and represent 7 % of the total.Ecotoxicological assays were performed using algae and arthropods by Avelelas et al., 2017 with LDH-ZnPT, LDH-CuPT, SiNC-ZnPT and SiNC-CuPT MCNMs, owing to their new anti-fouling properties.Moreover, ZnO-conjugated graphene oxide, ZnO-conjugated carbon nanotubes, TiO 2 -conjugated GO, and TiO 2 -conjugated CNT, developed to impart higher catalytic efficiency, were tested for their adverse effects on Thalassiosira pseudonana, chosen as a model for diatom physiology studies, being widely distributed throughout entire marine food chains (Baek et al., 2020).Recently, Klekotka et al., 2022 also investigated the effects of core-shell Fe 3 O 4 -Ag MCNMs (a bimetallic material which can be recovered and reused) on A. fischeri.
For several functionalized metal and metal oxide NMs (e.g., ZnO-and CuO-based NMs), the dissociation or disintegration processes involving the coating may lead to exposure of test organisms to undissolved and dissolved NMs, or even fraction of them (Di Cristo et al., 2021).For this reason, the distinction among these forms is essential for properly interpreting the results of ecotoxicological assays, calling for experimental data to provide information on the different transformations to which these NMs can undergo in the different test media.
Starting from 2016, another broadly investigated material is plastic, which can reach the marine environment due to its persistent and ubiquitous occurrence, and can be transformed into micro and nanoplastics after decomposition (Gonçalves and Bebianno, 2021).Among all the different plastic materials, the ecotoxicological effects of polystyrene (PS) nanoplastics have been the most studied ones (which correspond to 83 % of the total), both as pristine PS (Costa et al., 2023;Ren et al., 2023;Sendra et al., 2019;Wang et al., 2023;Yao et al., 2023) or by comparing the effects of pristine PS and PS modified with different functional groups, such as anionic carboxylates (-COOH), cationic amino (− NH 2 ) or sulfonic acid (-SO 3 H) groups (Bergami et al., 2016(Bergami et al., , 2017;;Manfra et al., 2017;Natarajan et al., 2020;Tallec et al., 2018;Varó et al., 2019; A. Brunelli et al.Y. Zhou et al., 2023).The effects of pristine PS were also compared to those from the mixture with PS, NMs or organic contaminants (Gonçalves and Bebianno, 2023;Wang et al., 2022).In addition to PS-based NMs, the other plastic NM tested was polymethylmethacrylate on marine microalgae and marine rotifer (Venâncio et al., 2019).

Tested marine species and exposure procedures
The different marine species tested towards NMs were grouped in Fig. 2 by their taxonomy levels: Bacteria, Algae, Rotifera, Arthropoda, Mollusca, Echinodermata and Chordata.In this classification, "other" refers to species that appeared only in one research article, i.e., the cnidaria Aurelia aurita, the anellidae Hediste diversicolor and the plant Halophila stipulacea.
Algae are the most studied marine organisms, especially the microalgae Phaeodactylum tricornutum and Dunaliella tertiolecta.The growth inhibition endpoint has been evaluated after 72 h of exposure for Phaeodactylum tricornutum (Bellingeri et al., 2022;Broccoli et al., 2021;Callegaro et al., 2015a;Clément et al., 2013;Peng et al., 2011;Prosposito et al., 2019;Sendra et al., 2019Sendra et al., , 2018) ) and after 24 to 96 h of exposure for Dunaliella tertiolecta (Bergami et al., 2017;Gambardella et al., 2015;Morelli et al., 2018Morelli et al., , 2013;;Schiavo et al., 2018).Easy cultivation, the relatively low cost of materials and equipment needed to complete a test, and the easy quantification of the growth endpoint favored using these organisms for testing all the NMs categories mentioned above, especially those with Ag-based NMs.
Lastly, effects on Echinodermata, Rotifera and Chordata were barely investigated, possibly due to the difficulties in performing the identification of the effects to complex organisms in parallel with the investigation of the NMs behaviour along the entire exposure duration, especially for long-term exposure tests (Skjolding et al., 2016).Regarding Echinodermata, effects on echinoids were the most explored, with the Mediterranean sea urchin Paracentrotus lividus being the Echinodermata most frequently tested (Gambardella et al., 2015(Gambardella et al., , 2013;;Rotini et al., 2018), followed by only one assay with the painted sea urchin Lytechinus pictus (Fairbairn et al., 2011).However, it should be underlined that the works dealing with adding NMs on echinoderm sperms/tissues were not considered in this review.As far as Rotifera, the works performed by (Clément et al., 2013;Manfra et al., 2017;Rotini et al., 2018;Venâncio et al., 2019) investigated the effects of NMs on Brachionus plicatilis under static conditions over an exposure period of 24 to 96 h.Effects on fish were tested on the following species: the sea bream Spaurus aurata, the killifish Fundulus heteroclitus and medaka fishes Oryzias latipes and Oryzias melastigma (Campbell et al., 2019;Barreto et al., 2020;Paterson et al., 2011;Wong and Leung, 2014).Regarding the exposure conditions maintained during the ecotoxicological assays, three procedures were used, i.e., static, semi-static and no static (aeration) exposure (Fig. 3).From the 89 papers examined, it emerged that static exposure was usually employed for Algae, Arthropoda, Bacteria, Echinodermata, Chordata and Rotifera due to the practical difficulties in changing the test media without affecting the exposed individuals.In contrast, semi-static exposure was preferred for Mollusca and the organisms within the "other" category.
The experimental set-up is of paramount relevance for testing NMs since a wide range of experimental conditions (e.g., the use of natural or artificial seawater, solvents, and dispersing or stabilizing agents) could alter the final ecotoxicological data and their comparability (Boros and Ostafe, 2020).Ad hoc guidelines/protocols have been developed by the OECD and the International Standard Organization (ISO) to overcome these flaws.However, only a few research articles followed the recommendation reported in these documents, including Bellingeri et al., 2022, who followed the ISO/TS 20787:2017 on the assessment of ecotoxicological effects of NMs in the Artemia sp., and Connolly et al., 2022, who followed the OECD 92:2000 for the assessment of biopersistent/ biodurable NMs including lysosomal membrane permeabilization (LMP).
Ultimately, choosing the appropriate test medium is another critical aspect for testing NMs with marine species.The Artificial Sea Water (ASW) medium required by ISO/OECD protocols helps to compare ecotoxicological data.At the same time, the Natural Sea Water (NSW) permits the assessment of more realistic scenarios, but the data obtained are less reproducible.Indeed, the chemical composition of seawater induces rapid NMs aggregation in ASW and Natural nano-filtered water, due to the high ionic strength and suppression of the electric double layer (EDL) on the NMs surface.Indeed, (Manfra et al., 2017) demonstrated that PS NMs were less toxic to the rotifer Brachionus plicatilis in NSW exposure media compared to ASW, probably due to the presence of dissolved organic matter, including colloids and proteins.
For microalgae testing, the most commonly used medium within this search is the Guillard's F/2, a mix of major nutrients, trace metals, and vitamins containing also chelating agents such as ethylenediaminetetraacetic acid (EDTA) (by Avelelas et al., 2017;Davarpanah and Guilhermino, 2019;Pikula et al., 2020;Poirier et al., 2018;Pretti et al., 2014;Sendra et al., 2018).However, chelating agents such as EDTA can induce morphology modifications of metal NMs, and consequent toxicological alterations (Melegari et al., 2019).Indeed, as proposed by Hund-Rinke et al., 2016 for freshwater ecotoxicological tests, an EDTA-free algal medium can be considered when metal NMs are assessed.Accordingly, Bellingeri et al., 2022 used a limited quantity of EDTA to assess the effects of Ag NMs on the diatom Phaeodactylum tricornutum following the recommendations of Leal et al., 2016, while a modified F/2 Guillard medium was also proposed by Hu et al., 2018.

Dispersion protocols and methods used
All the ecotoxicological studies examined in this review were grouped by the year of publication and divided in six categories (Fig. 4), as follows: i) papers in which a dispersion protocol or TG 318 listed in Table 1 was used to prepare a stock suspension and where the physicochemical characterization of the NMs in the exposure media of the assay was performed (black); ii) works employing a dispersion method developed ad hoc and in which the physico-chemical characterization of the NMs was investigated in the exposure medium of the assay & studies in which the NMs were already provided as a stable stock dispersion and characterized in the media (green); iii) studies where only a thorough physico-chemical characterization of the NMs in the exposure medium was conducted (light green); iv) works where only a dispersion method was used, without carrying out any physico-chemical characterization of NMs in the ecotoxicological media of interest (orange); v) studies in which only partial information on dispersion method and/or physicochemical characterization of NMs in ecotoxicological media during the assay were provided (light yellow); vi) no dispersion protocol/method and no physico-chemical characterization in ecotoxicological media during the assay was used (brown).The overall information on the parameters investigated and the techniques used to characterize the NMs in the testing ecotoxicological media are reported in Table S1.
Fig. 4 highlights that only very few authors followed a dispersion protocol: Sendra et al. (2017) and Calisi et al. (2022) followed one of those listed in Table 1, i.e., the protocols released by CEINT/NIST (special publication 1200 series) and the PROSPEcT protocol, respectively.Furthermore, Barrick et al., 2019 andConnolly et al., 2022 used other two protocols (i.e., the NANOGENOTOX protocol applied to disperse carbon nanofibers and the Nanosolutions project standard operating procedure dispersion protocol but not available online, respectively) not included in Table 1 but anyway displayed into the "dispersion protocol and characterization" category in Fig. 4.
Moreover, Fig. 4 shows that more than half of the studies analysed followed both a dispersion method and carried out the physicochemical characterization of NMs in the ecotoxicological media used for the Fig. 3. Exposure conditions used for marine organisms during the ecotoxicological assays analysed.
A. Brunelli et al. assays, while 15 % of the studies provide only partial information on the dispersion methods used and/or on the physico-chemical characterization of NMs in the ecotoxicological media.Lastly, the other studies fit into one of the other three categories (≤ 10 % each), in which only the dispersion method or characterization in ecotoxicological media was described or in which the starting stock dispersion was already stable.It should be remembered that, depending on the chemical characteristics of the sample considered and its stability, less invasive methods to disperse NMs, such as vortexing or mechanical mixing, can be an alternative to the sonication methods as, e.g., for nanoplastics or for MCNMs, avoiding unwanted breakages and allowing to mimic the natural conditions of the marine environment in a more realistic way (Bergami et al., 2017).

Remarks and conclusions
This review highlights a strong need to align different procedures to disperse NMs for ecotoxicological testing with the available international standard protocols, with the aim to obtain comparable results that can provide strong scientific evidence for the regulation and risk assessment of NMs in the marine environment.Existing standard dispersion protocols and technical guidelines for ecotoxicity testing of NMs have been recommended and, when not suitable, adaptations should be considered, possibly based on methods and procedures already used in the literature.Anyhow, if no standard dispersion protocol can be adopted, the experimental conditions used to obtain the stock dispersion should be clearly indicated, specifying the NMs' concentration, the medium composition, the type of sonication, the DSE, the composition and concentration of any dispersant (if needed), the maximum assured stability time and the techniques used.
As concerns other critical methodological aspects for the ecotoxicological testing of NMs in the marine environment, this review suggests the adoption of experimental conditions that allow to obtain a stable dispersion of NMs along the duration of the assay -depending on both the NM type and the test species -taking into account the processes of agglomeration and sedimentation of NMs due to the high ionic strength of marine water, which can influence the actual exposure concentration and thus affect the results of the assays.In this regard, a comprehensive physicochemical characterization of the stock dispersion as well as of the dilute dispersions used for ecotoxicity assays must be carried out, to gather essential information for the interpretation of the observed effects.This will allow to investigate the most important processes that NMs can undergo during assays, by considering the chemical composition of the test medium as well as the characteristics and the feeding

Fig. 1 .
Fig. 1.Relative abundance of NMs tested towards marine water organisms.Papers reporting more than one ecotoxicity assay are considered as separate studies.

Fig. 2 .
Fig. 2. Tested species grouped by taxonomy ("Other" include Cnidaria, Anellidae and vascular Plants) and by the different NMs tested.Articles reporting more than one ecotoxicity assay are considered as separate studies.

Fig. 4 .
Fig. 4. The 89 studies selected, grouped by years of publication and divided into the six categories displayed.Below the x axis, dispersion protocols and the TG 318 by years of publication are reported.
several protocols for in vitro toxicity tests have been developed since 2010 for dispersing metal oxide NMs and carbon nanotubes, such as NANOGENOTOX, ENPRA, NANO-IMMUNE, the CEINT/NIST 1200 series protocols (produced by the Duke University's Center for the Environmental Implications of Nanotechnology (CEINT) and the National Institute of Standards and Technology (NIST)).In addition, other protocols were generated within projects from the German BMBF sponsorship program as well as from other European research projects (available at https://nanopartikel.info/en /knowledge/operating-instructions/).More recently, the Harvard Dispersion Dosimetry Protocol (HDDP) by DeLoid et al., 2017 and the protocol by Kaur et al., 2017 were published.

Table 1
Available NMs dispersion protocols and guidelines for aquatic ecotoxicological testing.
b Ice-water bath during sonication is always recommended in each protocol listed.c Delivered Sonication Energy (DSE) [J/mL] = (P × t)/V, where P [J/s] = Delivered Acoustic Power = (dT/dt) × M × C p , T = temperature, t = time, M = mass, C p = specific heat, V = volume.